Abstract
Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.
Highlights
Maintenance of protein homeostasis, or proteostasis, is accomplished by the proteostasis network comprising biological pathways that control the rate of protein synthesis and the efficiency of protein folding, trafficking and degradation [1]
The fibrils formed from the three different proteins presented with the typical amyloid structure, as seen by transmission electron microscopy (TEM)
These two compounds are amyloid-specific dyes that change their spectroscopic behavior when bound to the cross-b fold present in amyloid fibrils [32,33]
Summary
Maintenance of protein homeostasis, or proteostasis, is accomplished by the proteostasis network comprising biological pathways that control the rate of protein synthesis and the efficiency of protein folding, trafficking and degradation [1]. The aggregation of peptides or proteins, exacerbated by aging, is genetically and pathologically linked to degenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease, and the systemic amyloid diseases [2]. A wide range of proteins, including those normally existing in a soluble folded state or as an intrinsically disordered monomer, can form cross-b-sheet amyloid fibrils owing to a mutation or because of environmental alterations [3]. Amyloid fibrils are made up of multiple interacting filaments, which are each comprised of thousands of monomers arranged at least as two-layer cross-b-sheets [4]. Because amyloid is stabilized by backbone H-bonding and side chain-side chain hydrophobic interactions, it has been proposed that any protein, regardless of its amino acid sequence, can form amyloid fibrils if subjected to appropriate solution conditions [6,7]
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